| Background and Objection:Prostate cancer is one of the most common cancer threat to men’s health. Prostate cancer remains the most commonly diagnosed malignancy and the second leading cause of cancer related deaths in men in the United States. In2004, about230,110cases of prostate cancer and29,900patients died of the disease. The incidence rate of prostate cancer is the third in men all over the world. In europe, each year about2.6million has been diagnosed prostate cancer accounts for11%of all male cancer, accounting for9%of all male cancer deaths..The incidence rate has been increasing year by year with the change of average life span and living condition in China. In1997the incidence rate of prostate cancer was2.0/100000male population, of4.55/100,000male population in2000, and it has been the third place malignant tumors of the male urinary tract and reproductive system.However, the pathogenesis of prostate cancer is not clear and clinical symptoms of prostate cancer is atypical. About1/3of patients can not be operated with the existence of local invasion or distant metastasis at the time of diagnosis, who is teated by androgen deprivation therapy. Unfortunately; this patients will be asandrogen-independent prostate cancer and (or) hormone-refractory prostate cancer after a median time of18to20months with the endocrine therapy. At present, it is a problem to treat advanced prostate cancer and it is still a hot topic of basic and clinical research how to effectively treat androgen-independent prostate cancer or hormone refractory prostate cancer. Therefore, to find effective molecular targeted has been a hot topic for the treatment of prostate cancer in recent years.Traditionally, it was believed that the only source of immunoglobulins (Igs) was mature B lymphocytes. However, some recent studies have demonstrated that malignant epithelial cells can also express Igs.Those investigators also detected transcription of the Ig gene in the same cell lines. It has been demonstrated that Ig can promote the growth and development of cancer. Babbage et al reported that secrete IgG of breast cancer cells can activate inducible citicoline Deaminase to enhance the occurrence of abnormal mutations in tumor cells. Golda et al had shown that in prostate cancer patient the serum IgG levels were usually elevated. Many study had been shown that Ig can play an important role in the process of cancer growth and development. Igs can also be produced by non-lymphoid lineage cells, rearranged Ig gene transcripts were identified in tumor cells, and the V-D-J region sequences revealed that IgG transcripts in tumor cells. Tumor-derived Ig can inhibit the effect of Ig mediated antibody-dependent cell-mediated cytotoxicity, play a certain role in promoting the proliferation of epithelial tumor cells. IgG was identified in a wide variety of soft tissue tumors and correlated well with proliferation markers and tumor grades. IgG may be a useful marker for cell proliferation in sarcomas. The blockade of tumor-derived IgG by either antisense DNA or antihuman IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro. More importantly, administration of antihuman IgG antibody also suppressed the growth of an IgG-secreting carcinoma line in immunodeficient nude mice. Prevalent expression of IgG in human carcinomas and its growth promoting functions may have important implications in growth regulation and targeted therapy of human cancers.The pathogenisis of prostate cancer is not clear, maybe it is close related to androgen. Some reported thar the serum IgG, IgA, or IgM antibodies concentration is usually increased in breast, lung, colon, ovarian cancer, liver cancer and prostate cancer patients. However, the detection of Ig expression and function in prostate cancer is rarely reported. It is still the lack of Ig expression of androgen dependent prostate cancer cell lines, and whether expression of Ig change from androgen-dependent prostate cancer cells to androgen-independent prostate cancer? Ig expression play a positive role in growth of tumor, Whether that promotes the proliferation of prostate cancer? Whether inhibited Ig expression can induce apoptosis in prostate cancer? If the prostate cancer can express IgG, it may provide new clues for prostate cancer carcinogenesis mechanism. It is a new method and research direction that to treat prostate cancer with molecular targeted therapy according to target of secretion IgG of cancer cell.It is the important point that how to search positive and effective targeted for the treatment of the prostate cancer, because the hormone-independent prostate cancer and refractory prostate cancer are not sensitive to a variety of endocrine therapy, radiotherapy and chemotherapy treatment and the median survival time is short. It has been provided a new approach for therapy of prostate cancer, which had been discovered some tumor-associated antigens such as prostate-specific membrane antigen (PSMA), niucin-1, cytotoxic T lymphocyte-associated antigen-4(CTLA-4). In recent years, according to cell signal target of drug, the molecular targetd therapy of prostate cancer involves mainly anti-vascular endothelial growth factor (VEGF), protein kinase inhibitor, targeted inhibition of epidermal growth factor receptor (EGRF) signaling pathway and the endothelin signal pathway antagonist, and so on. Protein networks of cellular signal transduction system is a complex, multi-factor cross-dialogue system. The most solid tumors are multi-target, multi-link regulation process. A single pathway targeted therapy is often sufficient to inhibit tumor progression owing to different stages of differentiation of tumor cells show significant heterogeneity. Considering traditional androgen deprivation single-target therapy, the treatment of prostate cancer and drug development will:be turned to the multi-target blocking, and to study the most critical signal transduction pathway of the hormone-refractory prostate cancer growth. It try to identify the most sensitive and specific therapeutic targets; to find out the most sensitive indicators that can predict the prognosis of prostate cancer and to achieve early diagnosis and early treatment.Nowadays, the technique of RNA interference(RNAi) becomes a new heated point of gene therapy and diagnostic techniques. RNA interference is a new credible method for inhibition of gene expression. RNAi is used to knockdown expression of gene targeted with small RNA(including small interference RNA,siRNA). If investigators transfer the chemosynthesis siRNA targeting some gene into tumour cells by liposomes or other assistant transfection methods, mRNA with homologous sequences would be degradation and the expression of gene targeted would be knocked down efficiently. RNAi has provided a new method for the treatment of prostate cancer. We envisage that RNA interference inhibit the expression of IgG, induced apoptosis of hormone-independent prostate cancer cell. It is explored for hormone-independent prostate cancer gene therapy.Objective:(1) To investigate the expression of immunoglobulin G (IgG) in human protate cancer cell lines (LNCaP and PC3) by flow cytometric and western blot and real-time quantitative PCR and to reveal the basic characteristics of PCa cells expressed Ig.(2) To investigate the clinic significance of the expression of immunoglobulin G (IgG) in protatatic carcinoma and to evaluate the correlation of IgG expression level and pathological grade in the prostate cancer tissue. To explore it whether become indicators of the malignancy degree of prostate cancer.(3) To construct and scree out the most effctive siRNA, transfect into prostatic carcinoma cell PC3, and to detect the effects of IGHG1knowdown on PC3cell growth and apoptosis. To explore a possible gene delivery strategy for prostate cancer gene therapy. To discuss the apoptosis mechanism in transfected siRNA PC3cell lines.Methods and materials:(1) Prostate cancer cell lines were cultured in RPMI1640. Flow cytometric was used to analyze expression of IgG in prtostate cancer cell lines. The expression level of IgG was tested by Real-time quantitative PCR (Q-PCR) and Western blot.(2) The expression level of IgG was detected by immunohistochemistry between prostate cancer tissues and benign prostate hyperplasia tissues. The correlation between IgG expression and clinicopathological features in prostate cancer tissues was analyzed.(3) The experiment included three groups:siRNA transfection, siRNA negative control, and blank control(with neither Lipofectamine2000nor siRNA). PC3cells were transfected with designed siRNA using the liposomes method, the expressions of IgG determined by Q-PCR and Western blot, and the proliferation and apoptosis of the PC3cells in three group detected by MTS and flow cytometry. Q-PCR and western blot were used to demonstrate caspase-3mRNA and protein expression level in three group after transfected siRNA.Results:(1) The positivity rates of IgG in LNCaP cells membrane and cytoplasm were 2.96%and89.22%respectively; The positivity rates of IgG in PC3cells membrane and cytoplasm were86.73%and90.99%respectively. There was significantly different expression level of IgG1mRNA between LNCaP and PC3cell (P=0.001). The IgG protein expression level of PC3cell was1.92±0.15, compared with that of LNCaP cell (1.05±0.86). There was significant difference of IgG protein level between in the PC3cell lines and the LNCaP cell lines (P=0.001).(2) Immunofluorescence histochemistry revealed that most of the collected prostate cancer and prostatic hyperplasia tissues expressed IgG in cytoplasma. There was obviously different expression of IgG between prostate Cancers(41/45) and prostatic hyperplasia (5/40)(P=0.000). There was positive relation between the IgG level in prostate cancer tissue and Gleason grade (It’s Spearman correlation coefficient rs=0.403, P=0.006).(3) The expression level of IgG mRNA (1.28±0.43) in the transfected PC3cells was decreased, with statistically significant difference from the blank control group (8.07±1.78) and negative control group (8.82±1.33)(P=0.002) by qPCR detected. The IgG protein expression level of the transfected group was1.24±0.11at48hours, markedly decreased as compared with that of the negative control(3.01±0.16) and blank control group(2.85±0.24)(P<0.001) with western blot analyzed. There were not significant difference between negative control group and blank control group, P>0.05.(4) The PC3cell growth was inhibited by RNAi with MTS test. There was significant differece between transfection group and blank control group (P<0.001). The PC3cell growth inhibition rates were31.3%and43.3%at48and72hours, respectively by MTS. The rate of apoptotic PC3cells were5.29%±0.41%in the experimental group significant differences higher compared with that of the blank group and the negative control group (1.49%±0.29%and1.92%±0.17%, respectively; P<0.01).(5) After48h transfected with siRNA, The expression level of caspase-3mRNA (3.93±0.28) in the transfected group was increased, with statistically significant difference from the blank control group (2.51±0.43) and negative control group (2.47±0.36)(P=0.004). The caspase-3protein expression level of the transfected group was1.34±0.11at48hours, markedly decreased as compared with that of the negative control(0.92±0.12) and blank control group(1.06±0.14)(P=0.014). There were not significant difference between negative control group and blank control group, P>0.05.Conclusions:(1) IgG is significantly expressed in LNCaP and PC3cells. High expression level of IgG in PC3cell is associated with development of prostatic carcinoma.(2) IgG is highly expressed in prostate cancer. The level of IgG in prostate cancer tissue is positive correlation with Gleason grade. The abnormal expression of IgG in prostate cancer plays a role in the occurring and development of prostate cancer and can be judged as a prostate cancer prognosis indicators.(3) The expression of IGHG1gene in PC3cell lines can be inhibited obviously both at mRNA and protein levels by the RNAi that targeting IGHG1. Target-silencing IGHG1gene can inhibited cell proliferation and induced apoptosis in PC3cell lines, the apoptotic cells were mainly at the early apoptotic stage. The effect of induing apoptosis in PC3cells after IGHG1silencd may mediated by caspase-3apoptotic singalin pathway. IgG can be further studied as an important target for protate cancer gene theray. |
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